The long-range goal of the study is to understand how reactive T-cells evade tolerogenic processes and contribute to autoimmunity. The specific goal of the application is to develop an existing transgenic mouse model (CD2Kb-4 mice) in which self MHC class I (H-2Kb)-specific thymocytes and T-cells evade tolerogenic processes and mediate skin graft rejection in young, but not in older mice. Two hypothesis will be tested using this unique model system. The central hypothesis posits that self peptide-specific CD4+ T-cells evade negative selection in thymus because neither direct nor indirect pathways of peptide presentation elicit negative selection of peptide-specific thymocytes when self proteins are presented exclusively by thymocytes themselves. The hypothesis will be tested by isolating H-2Kb-specific CD4+ and CD8+ T-cell clones elicited by skin grafts applied to CD2Kb-4 mice and determining their antigenic recognition specificities (Specific Aim 1). Whether H-2Kb in soluble form, prepared from cell lysates or expressed by intact cells elicits responses from these T-cell clones in vitro will be determined to evaluate whether peptides can be presented directly or indirectly to H-2Kb-specific T-cell clones (Specific Aim 2). A rigorous test of the central hypothesis will be conducted. Two new lines of T-cell receptor (TCR) transgenic mice using one CD4+ and one CD8+ T-cell clone as a source of productively rearranged TCR genes will be generated. TCR transgenic mice will be intercrossed with CD2Kb-4, and other existing H-2Kb transgenic mice expressing H-2Kb in various cell types to assess the effect of H-2Kb expression on thymocyte and T-cell development and function in double transgenic [TCR x H-2Kb] mice (Specific Aim 3). Using the same mice, the second hypothesis, that tolerance to skin grafts expressing H-2Kb is acquired as CD2Kb-4 mice grow older because direct and/or indirect presentation of H-2Kb-peptides is an ongoing tolerogenic process in vivo, will be tested. Specifically, whether mature T and B-cells expressing H-2Kb influence the tolerance status of naive H-2Kb-peptide specific T-cells over time, will be examined (Specific Aim 3). Finally, the basis of T-cell discrimination between donor skin grafts and host lymphocytes expressing H-2Kb will be examined. This goal will be achieved by immunizing [TCR x CD2Kb-4] mice with H-2Kb-peptides or with cells or skin grafts expressing H-2Kb and examining the T-cell response elicited using phenotypic and functional criteria (Specific Aim 4). The results from these studies will elucidate the contribution of direct and indirect presentation of self peptides derived from proteins expressed exclusively in lymphoid cells to tolerance induction in thymus and the periphery. The studies are also relevant to the unresolved debate concerning the mechanistic basis of T-cell clonal ignorance and to the contribution of indirect peptide presentation of self peptides to rejection of tissue allografts between MHC identical individuals.